The universality of calcium as an intracellular messenger depends on its enormous versatility. Cells have a calcium signalling toolkit with many components that can be mixed and matched to create a wide range of spatial and temporal signals. This versatility is exploited to control processes as diverse as fertilization, proliferation, development, learning and memory, contraction and secretion, and must be accomplished within the context of calcium being highly toxic. Exceeding its normal spatial and temporal boundaries can result in cell death through both necrosis and apoptosis.

The versatility and universality of calcium signalling

### A. Overview

Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface receptors termed purine receptors. In this review particular emphasis is placed on the discrepancy between the

/pdf/receptors-for-purines-and-pyrimidines-56f78tnpew.pdf

Receptors for Purines and Pyrimidines

FACS-optimized mutants of the green fluorescent protein (GFP)

To live or to die? This crucial question eloquently reflects the dual role of Ca2+ in living organisms--survival factor or ruthless killer. It has long been known that Ca2+ signals govern a host of vital cell functions and so are necessary for cell survival. However, more recently it has become clear that cellular Ca2+ overload, or perturbation of intracellular Ca2+ compartmentalization, can cause cytotoxicity and trigger either apoptotic or necrotic cell death.

Regulation of cell death: the calcium-apoptosis link.

Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy

Microdomains of high intracellular calcium ion concentration, [Ca2+]i, have been hypothesized to occur in living cells exposed to stimuli that generate inositol 1,4,5-trisphosphate (IP3). Mitochondrially targeted recombinant aequorin was used to show that IP3-induced Ca2+ mobilization from intracellular stores caused increases of mitochondrial Ca2+ concentration, [Ca2+]m, the speed and amplitude of which are not accounted for by the relatively small increases in mean [Ca2+]i. A similar response was obtained by the addition of IP3 to permeabilized cells but not by perfusion of cells with Ca2+ at concentrations similar to those measured in intact cells. It is concluded that in vivo, domains of high [Ca2+]i are transiently generated close to IP3-gated channels and sensed by nearby mitochondria; this may provide an efficient mechanism for optimizing mitochondrial activity upon cell stimulation.

https://science.sciencemag.org/content/sci/262/5134/744.full.pdf

Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria

Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells

Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

Ca2+ is a key regulator not only of multiple cytosolic enzymes, but also of a variety of metabolic pathways occurring within the lumen of intracellular organelles. Until recently, no technique to selectively monitor the Ca2+ concentration within defined cellular compartments was available. We have recently proposed the use of molecularly engineered Ca(2+)-sensitive photoproteins to obtain such a result and demonstrated the application of this methodology to the study of mitochondrial and nuclear Ca2+ dynamics. We here describe in more detail the use of chimeric recombinant aequorin targeted to the mitochondria. The technique can be applied with equivalent results to different cell models, transiently or permanently transfected. In all the cell types we analyzed, mitochondrial Ca2+ concentration ([Ca2+]m) increases rapidly and transiently upon stimulation with agonists coupled to InsP3 generation. We confirm that the high speed of mitochondrial Ca2+ accumulation with this type of stimuli depends on the generation of local gradients of Ca2+ in the cytosol, close to the channels sensitive to InsP3. In fact, only activation of these channels, but not the simple release from internal stores, as that elicited by blocking the intracellular Ca2+ ATPases, results in a fast mitochondrial Ca2+ accumulation. We also provide evidence in favor of a microheterogeneity among mitochondria of the same cells, about 30% of them apparently sensing the microdomains of high cytosolic Ca2+ concentration ([Ca2+]c). The changes in [Ca2+]m appear sufficiently large to induce a rapid activation of mitochondrial dehydrogenases, which can be followed by monitoring the level of NAD(P)H fluorescence. A general scheme can thus be envisaged by which the triggering of a plasma membrane receptor coupled to InsP3 generation raises the Ca2+ concentration both in the cytoplasm (thereby triggering energy-consuming processes, such as cell proliferation, motility, secretion, etc.) and in the mitochondria, where it activates the metabolic activity according to the increased cell needs.

/pdf/mitochondrial-ca2-homeostasis-in-intact-cells-1sdpk0efyu.pdf

Mitochondrial Ca2+ homeostasis in intact cells.

Nerve activity can induce long-lasting, transcription-dependent changes in skeletal muscle fibers and thus affect muscle growth and fiber-type specificity. Calcineurin signaling has been implicated in the transcriptional regulation of slow muscle fiber genes in culture, but the functional role of calcineurin in vivo has not been unambiguously demonstrated. Here, we report that the up-regulation of slow myosin heavy chain (MyHC) and a MyHC-slow promoter induced by slow motor neurons in regenerating rat soleus muscle is prevented by the calcineurin inhibitors cyclosporin A (CsA), FK506, and the calcineurin inhibitory protein domain from cain/cabin-1. In contrast, calcineurin inhibitors do not block the increase in fiber size induced by nerve activity in regenerating muscle. The activation of MyHC-slow induced by direct electrostimulation of denervated regenerating muscle with a continuous low frequency impulse pattern is blocked by CsA, showing that calcineurin function in muscle fibers and not in motor neurons is responsible for nerve-dependent specification of slow muscle fibers. Calcineurin is also involved in the maintenance of the slow muscle fiber gene program because in the adult soleus muscle, cain causes a switch from MyHC-slow to fast-type MyHC-2X and MyHC-2B gene expression, and the activity of the MyHC-slow promoter is inhibited by CsA and FK506.

https://www.pnas.org/content/pnas/98/23/13108.full.pdf

Marta Murgia

Papers

Microdomains with high Ca2+ close to IP3-sensitive channels that are sensed by neighboring mitochondria

Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells

Oxidized ATP. An irreversible inhibitor of the macrophage purinergic P2Z receptor.

Mitochondrial Ca2+ homeostasis in intact cells.

Calcineurin controls nerve activity-dependent specification of slow skeletal muscle fibers but not muscle growth